Combining an Enhanced Polyphosphate Kinase-Driven UDP-Glucose Regeneration System with the Screening of Key Glycosyltransferases for Efficient <i>In Vitro</i> Synthesis of Nucleoside Disaccharides

Nucleoside disaccharides are essential glycosides that naturally occur in specific living organisms. This study developed an enhanced UDP-glucose regeneration system to facilitate the <i>in vitro</i> multienzyme synthesis of nucleoside disaccharides by integrating it with nucleoside-specific glycosyltransferases. The system utilizes maltodextrin and polyphosphate as cost-effective substrates for UDP-glucose supply, catalyzed by α-glucan phosphorylase (αGP) and UDP-glucose pyrophosphorylase (UGP). To address the low activity of known polyphosphate kinases (PPKs) in the UDP phosphorylation reaction, a sequence-driven screening identified RhPPK with high activity against UDP (>1000 U/mg). Computational design further led to the creation of a double mutant with a 2566-fold increase in thermostability at 50 °C. The enhanced UDP-glucose regeneration system increased the production rate of nucleoside disaccharide synthesis by 25-fold. In addition, our UDP-glucose regeneration system is expected to be applied to other glycosyl transfer reactions.